1. Field of the Invention
The invention generally relates to plant biotechnology. More specifically, it relates to improved methods for transformation of monocots with a gene of interest.
2. Description of the Related Art
Genomics-based approaches in plant biotechnology have enabled identification and isolation of a large number of genes and have necessitated the need for reliable and efficient high-throughput transformation production systems for testing the utility of these genes by transforming them into economically important monocots such as corn. Agrobacterium-mediated transformation of monocots such as corn, rice, and wheat is a widely used experimental approach, often with the use of meristematic tissue such as immature embryos as the explants of choice (e.g. Ishida et al., 1996; Zhao et al., 2001; Frame et al., 2002). For rice, transformation of imbibed seeds has also been reported (Toki et al., 2006). To date, the most common methods following the contacting of cells with Agrobacterium include: culturing explant tissue such as immature embryos (“co-culture”), possibly including a “delay” or “resting” (non-selective) step, and followed by culture on selection medium containing auxin(s) allowing de-differentiation of cells to form callus. During this callusing phase, transformed resistant callus tissue is selected in the presence of an appropriate selection agent on a selection medium. This is followed by growth of cells under conditions that promote differentiation of the callus and regeneration of the callus into plants on regeneration and rooting media. This process has typically required at least 10-12 weeks to produce plants that can be transferred to soil for further growth. The process also requires several manual transfers of tissue throughout the transformation process and uses several different types of media.
Thus use of standard transformation and regeneration protocols is time consuming and inefficient, and negatively impacts the transgenic product development timeline, given that there is usually a seasonally limited “priority development window” for making decisions regarding which genetic constructs to prioritize for use in larger scale transformation work based on results obtained during initial research. There is therefore a need in the art of monocot transformation to produce transgenic plants quickly to provide more time and flexibility for making research and product development decisions during a priority development window. Such a high throughput system for corn transformation could produce a large number of transgenic plants for testing genes and creating useful plants while lowering material and labor costs.
Further, embryogenic culture responses of different breeding lines differ greatly, limiting the genotypes of crops such as corn that can be transformed. Accordingly, some lines can form embryogenic callus readily, although many, in general, fail to form any embryogenic callus. Such lines are often considered “recalcitrant” lines. This can require use of non-elite lines for transformation, which can require many generations of breeding to produce agronomically-elite transgenic varieties. Thus there is further a need for transformation methods that allow transformation of hitherto “recalcitrant” corn genotypes to allow a wider choice of transformable lines for product development, as well as for screening such genotypes for their potential transformability.